The lignocellulosic biomass resource represents a considerable renewable energy source and it is obtained from agricultural and forest residues or from wood transformation by-products, as well as dedicated crops (woody or herbaceous plants). The lignocellulosic material consists of three major elements interlinked in a complex network. These elements are cellulose, hemicelluloses and lignin. Their respective proportions vary depending on the exact origin of the biomass.
The procedure often recommended to obtain fermentescible sugars from cellulose is enzymatic hydrolysis. Degradation of cellulose to glucose requires the synergetic action of three categories of enzymes, cellulases, classified according to their activity:
the endoglucanases cut the cellulose randomly at the level of the amorphous zones of the cellulose,
the exoglucanases (or cellobiohydrolases when they produce cellobiose) act progressively on the free ends of the cellulose chains, releasing glucose or cellobiose,
the β-glucosidases (or cellobiases) hydrolize the soluble cellodextrines and the cellobiose to glucose.
One of the main barriers currently limiting the industrial development of this process is the high cost associated with the production of cellulases. In order to improve the activity of commercial enzymatic cocktails, supplementation of the cellulases with β-glucosidases is described in the literature, so as to annihilate the well-known inhibiting effect of cellobiose on the activity of endo- and exoglucanases.
In order to reduce the amount of enzymes used, various solutions have been proposed to recycle these enzymes. At the end of the enzymatic hydrolysis process, the enzymes are partly in free form in the hydrolysate and partly bonded to the solid residue.
In patent FR-B-2,608,625 filed by the applicant, a method consisting in recovering the major part of the enzymes is provided: the free enzymes are recovered by adsorption on the new substrate to be converted, whereas the bonded enzymes are used again by recontacting the solid residue with the new substrate. In cases where the cellulases are supplemented with β-glucosidases to improve the activity of the enzymatic cocktail, the method provided is however not satisfactory: after recycling, a significant activity loss is observed, associated with the accumulation of cellobiose (Ramos et al, Applied Biochemistry and Biotechnology, 1994, 45 (6), 193-207). In fact, this method does not allow to efficiently recycle the free β-glucosidases that have very little affinity with the lignocellulosic substrate.
Supplementation of commercial cellulases with supported β-glucosidases is a method described by Woodward et al. (“Use of immobilized beta-glucosidase in the hydrolysis of cellulose”, 1993, ACS symposium series, 533, 240-250) which allows to re-use several times the β-glucosidases without any activity loss or appreciable decrease in the conversion of cellulose to glucose. Immobilization of the β-glucosidases allows their stability to be notably improved: Aguado et al. (Biotechnology and Applied Biochemistry, 1995, 17(1), 49-55) showed that the immobilization of Penicillium funiculosum β-glucosidases on nylon powder allows to obtain a stable activity for 1500 min at 50° C., whereas the same enzymes in the free state deactivate from 40° C. on. Aspergillus niger β-glucosidases are stable in the free state up to 45° C., whereas in immobilized form on Eupergit C, their activity remains stable up to 65° C. (Tu et al, 2006, Biotechnology Letters, 28 (3), 151-156). On the other hand, at such temperatures, the cellulases show little stability and they rapidly deactivate, which impacts the hydrolysis efficiency.
Recycling the supported β-glucosidases requires extracting them from the reaction mixture containing a solid residue, which cannot be readily done when the cellulases and the β-glucosidases are used in the same reactor.
On the other hand, non-productive adsorption of the β-glucosidases on lignin is a known source of limitation of enzymatic hydrolysis. One known solution for limiting this adsorption consists in adding surfactants and/or proteins such as bovine serum albumin (BSA) (Yang et al., 2006, Biotechnology and Bioengineering, 94 (4), 611-617).
These main limitations can be raised by implementing the sweet juice production method according to the present invention.